Hallucinogen actions present a complicated picture because they (a) cause responses that differ from those of other agonists on specific targets such as 5-HT2 receptor subtypes (5-HT2A, 5- HT2C);(b) modulate multiple signaling pathways in specific neurons;and (c) involve several brain regions in their behavioral effects. In the previous period of this Program Project we have developed a multi-scale approach to this complex problem that connects insights about molecular and cellular signaling events to larger scale processes in the neurons and brain regions involved in the generation of the behaviors. We have also developed the powerful genetic animal models and the protein engineering constructs that can be used to probe the consequences of such specifically designed constructs in vitro and in vivo. The research plan of this PPG renewal application presents such a multi-scale investigation, based on an interdisciplinary protocol of iterative studies in the three Projects. The coordinated and integrated studies span scales of size and organization: from molecular structures of activated forms of the wild type and mutant GPCRs and their interactions in the signaling pathways, that are studied in Project 1;to the quantitative genomics-based approach termed Transcriptome Fingerprinting that reflects differential signaling responses through these receptors, and the identification of neurons and specific molecular mechanisms at the cell signaling level that are involved in hallucinogen-specific responses, pursued in Project 2;and onward to genetically modified mice with brain-region-specific manipulations of the 5-HT2AR and its interactions with other receptors and signaling mediators, in Project 3. New constructs of the receptors and their signaling-associated proteins, engineered in Project 1 from bioinformatics, structure-based analysis and simulations, will be expressed in Project 2 to (i) study of the cellular mechanisms underlying hallucinogen signaling specificity;(ii) test mechanistic hypotheses about receptor dimerization and interactions with scaffolding proteins generated in Project 1;and (iii) select the mechanistically most revealing ones for knock-in/knock-out and region-specific restoration and deletion experiments in Project #3, in order to elucidate the behavioral phenotypes associated with support or disruption of specific mechanisms. Through its integrative construct and focus, this research plan is expected to continue to have significant implications for understanding drug abuse in general, as well as on the design of new GPCR-based therapeutics for drug addiction. PROGRAM CHARACTERISTICS